Ophysio Inc. — Department of Health and Human Services SBIR Phase I: 400

Description

PROJECT SUMMARYHuman embryonic stem cellshEScrepresent a potentially unlimited supply of tissues for regenerative therapiesHowevercurrent differentiation methods result in heterogeneous populations of cells in which the desired product isat besta fraction of the totalMoreovercarry over undifferentiated cellsas well as cells that de differentiate upon transplantationhave been shown to form teratomas in animal modelsThe risk of teratomas is arguably the foremost obstacle towards the clinical implementation of hESc based therapiesand a major cause of concern for regulatory agenciesIn order to address this problemwe set out to engineer hESc with control mechanisms allowing for the selective ablation ofatumorigenic cellsandbcells differentiated along non desired fatesOur preliminary data indicate that a construct of our inventionstably integrated in the genome of the HhESc lineimparts such selectivity both in vitro and upon transplantation in vivoWe have chosen pancreaticcells as a model to test our hypothesisas hESc derivedlike cells are now entering clinical trials for typediabetesHoweverthe principle behind our strategy could be adapted to any cell type of choiceOur control mechanisms are based onathe activation of a suicide geneHSV TKin cells that resume continue tumorigenic proliferationandbthe irreversible inactivation of a second suicide geneNTRin cells that express insulinOur central hypothesis is that hESc lines where these constructs are targeted to asafe harborgenomic locationthe ROSAlocuswill work as effectively as they do when their integration is randomas per our preliminary dataWe plan to test this hypothesis by pursuing the following specific aimsTo integrate the above constructs into the ROSAlocus of a human hESc line by CRISPR Casgene editing techniquesandTo perform in vivo tests of function in rodentsThe proposed research is innovative because it provides a degree of selectivity that is lacking in conventional suicide gene based strategiesin which activation of the transgene typically brings about the destruction of the entire graftWe anticipate a positive impact of our research on the fieldbecause such cell lines would be ideally suited for the development and safe clinical implementation of differentiation protocolsOur researchthereforeis designed to break barriers that stand in the way of the widespread clinical use of hESc and will pave the ground for a subsequent Phase II proposal aimed at the generation of cGMP grade hESc with this targeted modification for safe use in the clinical practice NARRATIVEIn order to realize the clinical potential of human embryonic stem cellshEScmethods to overcome both the relatively high incidence of tumorsteratomasand the low efficiency of differentiation must be devisedHere we propose a double fail safe mechanism to address these two problemshESc engineered with a construct of our invention will activate a suicide gene in tumorigenic cells and de activate a second in cells of the desired characteristicsOur proposal thus responds to the general mission of the NIHas a positive outcome will speed up the safe and efficacious translation of hESc research into clinical therapies